Dual function of the alcohol dehydrogenase of Drosophila melanogaster: ethanol and acetaldehyde oxidation by two allozymes ADH-71k and ADH-F

Abstract

Until recently the alcohol dehydrogenase of Drosophila melanogaster was thought to act only in the first step of primary alcohol oxidation, producing an aldehyde. Instead, acetic acid is the main product of a two-step process. A rapid procedure was developed for the isolation and purification of two allozymes. The thermostability of the purified enzymes was found to be very different, t 1/2 at 35 degrees C, being 45 min and 130 min for ADH-F and ADH-71k respectively. The kinetic parameters of ethanol oxidation by the two purified allozymes were determined within physiological substrate and coenzyme ranges. The use of artificial electron acceptors has a notable influence on the ethanol oxidation: the apparent Michaelis constants increase; the oxidation rate with ADH-71k increases, whereas it decreases with ADH-F. Purified ADH is shown to be able to catalyze the oxidation of acetaldehyde solely in the presence of NAD+, and PMS and MTT as artificial electron acceptors. From the kinetic data the relative in vivo oxidation rates of ethanol by both ADH allozymes were calculated. ADH-F turned out to be somewhat less effective (30%-40%) than ADH-71k. The physiological consequences of these differences are discussed.

Kinetics and thermodynamics of ethanol oxidation catalyzed by genetic variants of the alcohol dehydrogenase from Drosophila melanogaster and D. simulans

“…Such data suggest functional differences in biochemical properties between the ADH variants. In vitro activities of fly extracts toward ethanol show a rank order of ADH-71k > ADH-F > ADH-S > simulans-ADH [4,11,13,14]. This rank order has been substantiated from in vivo studies in third-instar larvae [12].…”

“…ADH protein was purified according to the affinity procedure given by Eisses et al [14]. In brief, about 5 g freshly frozen adult flies (age 5-10 days) were hand-homogenized in a mortar by means of a pestle in 50 ml 20 mM Tris-HC1 buffer, 5 mM MgC1 z, 0.4 mM EDTA, pH 6.4 (buffer A).…”

“…The question remained whether activity differences originate from differences in protein quantity in vivo and/or in kinetic-catalytic properties [12,15]. Previous kinetic studies on purified ADHs from Drosophila at a fixed temperature under optimum pH conditions have demonstrated differences in kinetic properties [11,14,16,17]. However, the enzyme proteins function at a presumed physiological pH of about 7 [5], whereas Drosophila experiences a temperature range of 15-35 ° C [18].…”

Functional significance of regulatory map and structural Amy variants in Drosophila melanogaster

“…melanogaster (Heinstra et aL, 1983;Bodmer and Ashburner, 1984;Eisses et al, 1985;Moxon et a!., 1985;Winberg et a!., 1985).…”

Participation of Drosophila melanogaster alcohol dehydrogenase (ADH) in the detoxification of 1-pentene-3-ol and 1-pentene-3-one

“…ADH enzymes catalyze the oxidation of primary and secondary alcohols into aldehydes and ketones, respectively. However, while aldehydes are further metabolized by means of an aldehyde dehydrogenase (David et al, 1984) and/or the ADH itself (Heinstra et at., 1983(Heinstra et at., , 1986bEisses et al, 1985;Moxom et a!., 1985), ketones seem to be metabolic dead ends (David et a!., 1981;Heinstra eta!., 1986a). In fact, primary alcohols can be used by Drosophila as food, while secondary ones cannot (van Herrewege et a!., 1980;Sánchez-Cañete et a!., 1986).…”